The overall goal of the proposed work is to examine the effect of proteolytic enzymes on normal and transformed cells in order to define the possible regulatory role of proteases. The experimental approach is twofold: 1. the addition of purified exogenous proteases to normal cells in order to biochemically analyze the mitogenic action of proteases and their ability to alter cellular behavior, 2. the study of endogenous proteases in malignantly transformed cells in order to ascertain their role in the altered phenotypic properties of transformed cells. In the first approach, the enzyme thrombin is used to stimulate resting chick enbryo fibroblasts into DNA synthesis. Besides examining the early biochemical alterations induced by thrombin, we have been studying the binding and internalization of thrombin using high specific activity radiolabeled thrombin. Preliminary results have indicated that a correlation exists between the internalization of thrombin and its mitogenic ability. In the second approach plasminogen activator (PA) is being studied in order to determine if it directly or indirectly effects the properties of chick embryo fibroblasts transformed by Rous sarcoma virus. PA is found elevated in malignant cells and like thrombin is a neutral serine protease having arginine specificity. A membrane-associated form of PA has been isolated and is presently being characterized. The extracellular, soluble form of PA is being purified in order to eventually obtain an antibody to PA. A purified form of PA and an antibody to it will prove to be valuable in defining the role of PA.